Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine.
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The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction. (+info)
Interaction of fMet-tRNA(fMet) with the C-terminal domain of translational initiation factor IF2 from Bacillus stearothermophilus.
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Analytical ultracentrifugation studies indicated that the C-terminal domains of IF2 comprising amino acid residues 520-741 (IF2 C) and 632-741 (IF2 C-2) bind fMet-tRNA with similar affinities (K(d) at 25 degrees C equal to 0.27 and 0.23 microM, respectively). Complex formation between fMet-tRNA(fMet) and IF2 C or IF2 C-2 is accompanied by barely detectable spectral changes as demonstrated by a comparison of the Raman spectra of the complexes with the calculated sum of the spectra of the individual components. These results and the temperature dependence of the K(d) of the protein-RNA complexes indicate that complex formation is not accompanied by obvious conformational changes of the components, and possibly depends on a rather small binding site comprising only a few interacting residues of both components. (+info)
Substitution of the heme binding module in hemoglobin alpha- and beta-subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin.
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In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions. (+info)
Intracellular reactions in single human granulocytes upon phorbol myristate acetate activation using confocal Raman microspectroscopy.
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We have obtained new evidence for the occurrence of intracellular NADPH-oxidase activity in neutrophilic and eosinophilic granulocytes upon stimulation with phorbol myristate acetate (PMA). PMA activation leads to a partial translocation of cytochrome b(558) from the membranes of the specific granules to the plasma membrane. It was suggested that NADPH-oxidase activity only takes place in the plasma membrane, leading to an extracellular release of oxygen metabolites because cellular self-destruction can be avoided in this way. The effects of PMA activation were indirectly studied in recent experiments employing scavengers of extracellular superoxide anion and hydrogen peroxide, and support for intracellular NADPH-oxidase activity was obtained. In this paper we use Raman microspectroscopy as a direct method to study intracellular molecular reactions that result from cellular triggering by PMA. The molecular specificity of this microscopic method enables us to show that intracellular reduction of both myeloperoxidase (MPO) and cytochrome b(558) occurs in neutrophilic granulocytes. Control measurements with cytochrome b(558)-deficient neutrophilic granulocytes did not show a reduction of intracellular MPO. This is direct support for the occurrence of intracellular NADPH-oxidase activity in organelles that must be in close contact with the azurophilic granules that contain MPO. Furthermore, a comparison was made with chemical reactions occurring in eosinophilic granulocytes after activation with PMA. Moreover, in these cells an intracellular reduction of eosinophil peroxidase was observed. (+info)
Resin content in cement liquids of resin-modified glass ionomers.
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Qualitative and quantitative analyses were conducted on four kinds of resin-modified glass ionomer (RMGI) cement liquids, LC, LC II, LC III (hereinafter referred to as LCs) and VM, using HPLC and laser Raman spectroscopic methods. HPLC revealed that among the RMGI liquids LCs contain 31-32% HEMA (2-Hydroxyethyl methacrylate), and VM contains 18% of the same. The composition of RMGI cement liquids varied significantly between manufacturers. In Raman spectroscopic analyses, the spectra of liquids of various ratios of polyacrylic acid and HEMA were measured, and calculations were made on the peak intensity ratios of C=C stretch vibration to C=O stretch vibration, common in both HEMA and polyacrylic acid. The composition ratio of polycarboxylic acid to HEMA of commercial glass ionomer cements was assessed by the regression curve generated by a combination of peak intensity ratios and composition ratios. In addition, Raman spectroscopy was able to identity the differences in form of the methacryloyloxy group. (+info)
Resonance Raman spectroscopy of sensory rhodopsin II from Natronobacterium pharaonis.
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Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485). (+info)
Pigment binding site properties of two photosystem II antenna proteins. A resonance raman investigation.
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Two light-harvesting proteins associated with photosystem II of higher plants, namely the major antenna complex LHCIIb and the minor Lhcb4 protein (CP29), have been investigated by resonance Raman spectroscopy. One of the two chlorophylls b and up to five of the six chlorophylls a present in Lhcb4 are shown to adopt similar binding conformations to the (presumably) corresponding molecules in LHCIIb, whereas at least two chlorophylls in the former protein assume unique conformations relative to the bulk complex. The overall conformation of bound xanthophyll molecules is identical in the two antenna proteins, although some small differences are apparent. The pigment binding properties of these two LHCs are discussed, with particular reference to possible structural motifs within this extended family of proteins. (+info)
Curvature properties of novel forms of phosphatidylcholine with branched acyl chains.
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We studied the properties of a series of phosphatidylcholine molecules with branched acyl chains. These lipids have previously been shown to have marked stimulatory effects on the side-chain cleavage activity of cytochrome P450SCC (CYP11A1), an enzyme of the inner mitochondrial membrane. The synthetic lipids used were diacyl phosphatidylcholines with the decanoyl, dodecanoyl or tetradecanoyl chain having a hexyl, octyl or decyl straight chain aliphatic branch at the 2-position. All three lipids lowered the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine, the lipids with longer acyl chains being more effective in this regard. As pure lipids all of the forms were found by X-ray diffraction to be predominantly in the hexagonal phase (HII) over the entire temperature range of 7-75 degrees C. The properties of the HII phase were unusual with regard to the small size of the lattice spacings and the small temperature dependence of the spacings. We used tetradecane to relieve hydrocarbon packing constraints to determine the intrinsic radius of curvature of the lipid monolayer. The elastic bending modulus was measured in the presence of tetradecane by introducing an osmotic gradient across the hexagonal phase cylinders with aqueous solutions of poly(ethylene glycol). The elastic bending modulus was found to be higher than that observed with other lipids and to increase with temperature. Both the small intrinsic radius of curvature and the high elastic bending modulus indicate that the presence of these lipids in bilayer membranes will impose a high degree of negative curvature strain. (+info)